Glutamate oxidase mutant with improved enzyme activity and application of glutamate oxidase mutant in production of alpha-ketoglutaric acid

Abstract

The invention belongs to the field of bioengineering, and particularly discloses a glutamate oxidase mutant with improved enzymatic activity. According to the invention, site-specific mutagenesis and saturation mutation are carried out on glutamate oxidase coding genes derived from streptomyces mobaraensis, wherein mutations are C536R, P532R, Q378D, V548E and G236D. After the mutant obtained by the invention is induced and purified, the specific enzyme activity of the optimal glutamate oxidase mutant is 1.69 U / mg and is improved by about 30% compared with that of wild bacteria, which shows that the 378-site amino acid residue has great influence on the catalytic action and stability of the enzyme. In addition, the invention also discloses a construction method of the glutamate oxidase mutant with improved enzymatic activity and application of the glutamate oxidase mutant in production of amino acid derivatives such as alpha-ketoglutaric acid. Therefore, the beneficial mutant provided by the invention can lay a good foundation for industrial fermentation production of alpha-ketoglutaric acid and other products.

Description

Technical Field

[0001] The invention relates to the construction of a glutamate oxidase mutant with improved enzyme activity and its application, and belongs to the technical field of genetic engineering. Background Art

[0002] α-ketoglutarate (α-KG) is an important organic acid and one of the key intermediates in the tricarboxylic acid cycle and amino acid metabolism. As a precursor of various amino acids and heterocyclic compounds, it plays an important role in cell metabolism, such as being a source of L-glutamine, L-glutamic acid and L-proline, as well as various metabolic intermediates. It not only participates in the synthesis and metabolism of amino acids, vitamins and organic acids in organisms, but also plays an important role in protein synthesis, skeletal muscle development and metabolism. Therefore, it is used to reduce the body loss of postoperative patients and long-term patients. α-KG also has anti-cyanide effects, scavenges free radicals, and delays aging. It is clinically used to reduce uremia in hemodialysis patients, promote wound healing, and treat chronic renal failure in hemodialysis patients. Due to its important physiological effects and excellent chemical properties, α-KG is widely used in food, medicine, cosmetics and other fields. Therefore, it has broad application prospects.

[0003] At present, there are three methods for the production and preparation of α-KG: chemical synthesis, microbial fermentation and biotransformation. The disadvantages of using chemical methods to produce α-KG are harsh conditions, high energy consumption and low yield. A large amount of harmful reagents such as cyanide, heavy metal ions, strong acids and alkalis are used in the synthesis process, and there are often serious food safety risks and environmental pollution problems. The fermentation method for producing α-KG has the advantages of abundant raw materials, low cost and high yield, but due to the excessive production of by-products such as pyruvic acid, fumaric acid and malic acid during the fermentation process, there are also disadvantages such as lack of strain resources, difficulty in separation and purification and long production cycle. At present, there are many studies on the use of Loya lipolytica for the production of α-KG by microbial fermentation, but due to its long fermentation cycle, low acid production rate, high by-products, difficult operation, high price and limited supply of normal alkanes, its industrial application is limited. The biotransformation method is simple to operate, mild conditions, high raw material utilization and high conversion rate. It has the advantages of easy separation and purification, and has important application needs in greatly saving production costs and solving the problem of excess glutamic acid production capacity. Therefore, this method is widely used in the production of α-KG.

[0004] Glutamate oxidase (LGOX) is a flavin protease with flavin adenine dinucleotide as a cofactor. It has high substrate stereoisomer selectivity, high catalytic efficiency, mild reaction conditions, and can oxidize L-glutamate to produce α-KG, hydrogen peroxide and ammonia without adding exogenous cofactors. L-glutamate oxidase has strict substrate specificity for L-glutamate and does not require any exogenous cofactors to catalyze glutamate, which makes it a great advantage as a catalyst for the production of α-KG. At present, a variety of L-glutamate oxidases have been isolated from Streptomyces, but most of them focus on the study of their protein structure and mechanism of action. The industrial application of L-glutamate oxidase for biocatalytic production of α-KG is still relatively small. Therefore, obtaining glutamate oxidase with excellent catalytic performance will be the key method to solve the problem of high yield of α-KG. Improving the catalytic activity of glutamate oxidase by using strategies such as random mutation and site-directed mutation is of great significance to improving the prospects for the industrial application of glutamate oxidase. Summary of the invention

[0005] The primary purpose of the present invention is to provide a glutamate oxidase mutant, so that its catalytic performance is improved, which is conducive to the production of metabolites such as α-KG. Another technical problem to be solved by the present invention is to provide a method for constructing and screening the above glutamate oxidase mutant.

[0006] The present invention is achieved through the following technical ideas: using a wild-type glutamate oxidase gene from Streptomyces mobaraensis as a template, designing primers for site-directed saturation mutagenesis, obtaining a mutant library through site-directed mutagenesis, then constructing a recombinant plasmid library with pET21b as a skeleton, transforming and introducing it into Escherichia coli BL21 (DE3), establishing a high-throughput screening system for the mutant library, and finally screening and obtaining glutamate oxidase mutants with improved enzyme activity through 96-well plate primary screening and test tube and shake flask rescreening.

[0007] A glutamate oxidase mutant is obtained by having one or both mutations of P532R or Q378D on the basis of wild-type glutamate oxidase, wherein the amino acid sequence of the wild-type glutamate oxidase is shown in SEQ ID NO: 3. The specific enzyme activity of the mutant can be increased by about 30% compared with the wild-type enzyme, for example.

[0008] The wild-type glutamate oxidase gene coding sequence provided by the present invention is shown as SEQ ID NO: 1, and the coding gene of the glutamate oxidase mutant, preferably, has a nucleotide sequence as shown in SEQ ID NO: 2.

[0009] The present invention further provides a recombinant expression vector containing the gene, preferably, it is a prokaryotic expression vector, including but not limited to pET21b and pXMJ19, etc. The present invention also provides a recombinant cell containing the encoding gene or the recombinant expression vector, preferably, it is a prokaryotic cell, including but not limited to Escherichia coli and Corynebacterium glutamicum, etc.

[0010] Finally, the present invention also provides the use of the mutant or its encoding gene in the biosynthesis of α-KG. Specifically, it includes the steps of culturing recombinant cells containing the encoding gene to produce α-KG, and collecting the produced α-KG. Optionally, it also includes the step of purifying the produced α-KG.

[0011] The beneficial effect of the present invention is that by conducting directed evolution and screening on L-glutamate oxidase from Streptomyces moyuanensis, a mutant of L-glutamate oxidase with improved catalytic activity is obtained. Therefore, the glutamate oxidase mutant provided by the present invention can lay a good foundation for efficient and rapid fermentation production of α-KG, and has better prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of α-KG concentration determination based on color change. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings, but it should not be construed as limiting the present invention. The experimental methods used in the examples are conventional methods well known to those skilled in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0014] Example 1 Construction of glutamate oxidase mutants L-glutamate oxidase (LGOX) can catalyze L-glutamate to produce α-KG. It is a flavin enzyme with flavin adenine dinucleotide (FAD) as a coenzyme, but because it contains a non-covalently bound FAD, it can complete the catalytic reaction without the addition of exogenous auxiliary factor FAD. Studies have found that this enzyme mainly exists in Streptomyces, but the enzyme production capacity varies greatly. Discovering and identifying glutamate oxidases with high activity or substrate specificity, and obtaining enzyme catalytic systems that meet the requirements of physiological research and production applications will provide favorable conditions for the industrial production of α-KG.

[0015] Protein language models have been proven to be effective in generating functional proteins and promoting the optimization of given proteins. ESM (Evolutionary Scale Modeling) is a method that uses deep learning technology to predict protein structure and function, which can easily predict and generate proteins [Meier J, et al. Language models enablezero-shot prediction of the effects of mutations on protein function. Advances in neural information processing systems, 2021, 34: 29287-29303.]. The core idea of ​​ESM is to regard the protein sequence as a language, each amino acid as a character, and then use an autoregressive neural network Transformer to learn the statistical laws of this language, and learn the evolutionary laws of proteins and the relationship between sequence-structure-function on a large-scale protein sequence database. The advantage of ESM is that it can extract rich information from protein sequences and predict the mutation effects of proteins, that is, the effects of changes in single or multiple amino acids in the protein sequence on the structure and function of the protein, such as stability, activity, affinity, etc. In the present invention, the protein language model and machine learning algorithm are used to predict the potential mutation sites and effects in the glutamate oxidase LGOX protein from Streptomyces moyuanensis (sequence as shown in SEQ ID NO: 3), and the five mutation sites with the highest ranking scores are selected for subsequent experimental verification, namely C536R, P532R, Q378D, V548E and G236D.

[0016] Primers LGOX-For / LGOX-Rev were designed based on the sequence, and the codon-optimized Streptomyces glutamate oxidase synthesized by GenScript was used as a template to obtain the wild-type glutamate oxidase gene fragment, which was subcloned into the pET21b plasmid backbone after NdeI / XhoI double digestion to obtain the recombinant plasmid pET21b-LGOX. A site-directed mutagenesis strategy was used to design mutation primers based on the amino acid sites to be mutated, and the recombinant plasmid pET21b-LGOX was used as a template to obtain a plasmid vector containing the mutation site by PCR, and transformed into the Escherichia coli expression strain BL21 (DE3).

[0017] Example 2 Activity determination of glutamate oxidase mutants The recombinant E. coli containing the pET21-LGOX, pET21-LGOX-C536R, pET21-LGOX-P532R, pET21-LGOX-Q378D, pET21-LGOX-V548E and pET21-LGOX-G236D plasmids obtained above were induced to express proteins. The specific method was as follows: single clones were picked and inoculated into 100 mL LB liquid medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl) for overnight culture. Subsequently, according to the starting OD 600 ≈0.1. Transfer the seed solution to 100 mL LB liquid medium and culture until the bacterial concentration OD 600 When the pH value reaches about 0.6-0.8, add IPTG at a final concentration of 0.4-0.6 mM to induce the expression of the target protein, adjust the culture temperature to 25-30°C, and continue the induction culture for 16-20 hours. After the induction is completed, the bacterial solution is subjected to high-pressure homogenization and crushing, and the laboratory standard Ni-NTA nickel column purification method is used to obtain the pure protein of glutamate oxidase. The protein concentration is determined by the Bradford method, that is, Coomassie Brilliant Blue G-250 detection solution is used to react with the protein, and bovine serum albumin is used as the standard.

[0018] The enzyme activity was determined by enzymatic reaction using pure enzyme obtained after purification by Ni-NTA nickel column. The total volume of the enzymatic reaction was 1 mL, including 600 μL 100 mM sodium glutamate substrate, 300 μL ddH2O and about 50 μL of appropriate concentration enzyme solution. After mixing, react at 37°C for 30 min and terminate the reaction by boiling in a boiling water bath for 10 min. After the reaction was completed, 20 μL of the enzymatic reaction solution was taken, 400 μL 2 mM 2,4-dinitrophenylhydrazine solution was added, incubated at 37°C for 20 min, and then 1 mL 1 M sodium hydroxide solution was added to terminate the reaction. The color developing solution was diluted 3 to 10 times and the absorbance value was measured (detection wavelength 390 nm), and the sample concentration was calculated by drawing a standard curve. The enzyme activity is defined as the amount of enzyme required to generate 1 μmol of α-KG per minute is 1 U.

[0019] The results showed that compared with the wild-type natural enzyme, the activity of the most preferred glutamate oxidase mutant LGOX-Q378D reached 1.69 U / mg, which was 30% higher than that of the wild-type bacteria. The activity of the second preferred mutant glutamate oxidase LGOX-P532R was 14% higher than that of the wild-type bacteria. However, the catalytic activity of other glutamate oxidase mutants was weaker than that of the natural enzyme to varying degrees.

[0020] Example 3 Construction and screening of glutamate oxidase Q378D site saturation mutations In view of the important contribution of the amino acid residue at position 378 of glutamate oxidase to the catalytic effect of the enzyme, a site-directed saturation mutagenesis strategy was adopted for the most preferred glutamate oxidase mutant LGOX-Q378D in Example 2, and random degenerate primers Q378-For (cgccatgttNNNgtgagcccgctgatgagctatg) and Q378-Rev (gcgggctcacNNNaacatggcgcagaccggtaaac) were designed, wherein N represents any of the four bases A, C, G or T, and a recombinant plasmid library containing saturation mutations at this site was obtained by PCR. The recombinant plasmid library containing specific site saturation mutations was introduced into the E. coli expression strain BL21 (DE3) for screening. The specific steps were as follows: a sterilized toothpick was used to pick up the transformant and added to a 96-well plate containing 200 μL LB medium. After culturing at 37°C and 800 rpm / min for 6-8 h, 100 μL was transferred to a 96-deep-well plate containing 600 μL LB medium, 0.4 mM isopropylthiogalactoside IPTG inducer was added, and the inducer was induced overnight at 16°C and 800 rpm / min for 14-16 h. After the induction was completed, the 96-deep-well culture plate was centrifuged at 4000 rpm / min for 10 min to obtain the bacterial cells, and the supernatant was discarded. After resuspending with a spray gun, 600 μL 1M sodium glutamate substrate was added to each well, and the whole-cell catalytic reaction was carried out in a well plate shaker at 37°C for 2 h. α-KG is easily reacted with 2,4-dinitrophenylhydrazine to generate reddish brown phenylhydrazone nitroquinone, and excellent mutants of glutamate oxidase are screened according to the color depth. In this example, after multiple rounds of saturation mutation and screening, no mutants better than LGOX-Q378D were found, suggesting that mutating the 378th glutamine in the glutamate oxidase encoding gene sequence to aspartic acid is the best solution.

[0021] Example 4 Application of glutamate oxidase mutants in bioconversion production of α-KG This example is used to provide an application of a glutamate oxidase mutant in the production of α-KG. In the specific implementation of this example, the preferred commercial expression vectors are pET21b and pXMJ19, and the preferred expression host bacteria are Escherichia coli ( Escherichia ) and Corynebacterium ( Corynebacterium ), more preferably Escherichia coli ( Escherichia coli ) and Corynebacterium glutamicum ( Corynebacterium glutamicum ), most preferably Escherichia coli BL21 (DE3) and Corynebacterium glutamicum ATCC 13032.

[0022] The pET21b-LGOX-Q378D recombinant plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and the Escherichia coli α-KG production strain was obtained after plate resistance screening. Primers were designed according to the sequence, and the glutamate oxidase mutant gene LGOX-Q378D was subcloned into the pXMJ19 backbone by molecular assembly technology such as one-step efficient seamless cloning (ClonExpress®II One Step Cloning Kit, Vazyme Biotech, China) to obtain the pXMJ19-LGOX-Q378D recombinant plasmid, which was transformed into Corynebacterium glutamicum ATCC 13032, and the Corynebacterium glutamicum α-KG production strain was obtained after plate resistance screening.

[0023] The α-KG bioconversion system was established using a fermentation tank, and a final concentration of 200 g / L of sodium glutamate monohydrate (MSG) substrate was added, 20 g / L of bacterial cells were added, the fermentation tank speed was controlled at 300-500 r / min, the dissolved oxygen was set at 10-30%, the catalytic reaction temperature was 30-37°C, and the catalytic reaction time was 20-40 h. After the reaction was completed, the components of the catalytic reaction liquid were quantitatively analyzed by liquid chromatography.

[0024] According to the analysis of liquid chromatography, the components of the whole cell conversion liquid are relatively simple, and there are few substrates and impurities. The engineered strains constructed based on the glutamate oxidase mutants show better α-KG production capacity. After 32 hours of catalytic reaction, the α-KG production of Escherichia coli α-KG production can reach 142.2 g / L, and after 24 hours of catalytic reaction, the α-KG production of Corynebacterium glutamicum α-KG production can reach 145.4 g / L. Compared with the recombinant engineered bacteria containing the unmutated glutamate oxidase gene, it has better advantages in whole cell catalytic time, product yield and production capacity, which can greatly save production costs and simplify the process flow, and has good prospects for industrial application.

Claims

1. A glutamate oxidase mutant, characterized in that: The wild-type glutamate oxidase has one or both mutations Q378D and P532R, wherein the amino acid sequence of the wild-type glutamate oxidase is shown in SEQ ID NO:

3.

2. The gene encoding the glutamate oxidase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant expression vector containing the coding gene as claimed in claim 2 or 3, preferably, it is a prokaryotic expression vector, including but not limited to pET21b and pXMJ19, etc.

5. A recombinant cell containing the coding gene according to claim 2 or 3 or the recombinant expression vector according to claim 4.

6. The recombinant cell according to claim 5, characterized in that It is a prokaryotic cell, including but not limited to Escherichia coli and Corynebacterium glutamicum.

7. Use of the glutamate oxidase mutant or the gene encoding it as claimed in claim 1 in the biosynthesis of α-KG.

8. The use according to claim 7, characterized in that The method comprises the steps of culturing the recombinant cells containing the encoding gene to produce α-KG, and collecting the produced α-KG.

9. The use according to claim 8, characterized in that The process also includes a step of purifying the produced α-KG.

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